The invention relates to a multi-enzyme product with glucoamylase, proteolytic and xylanase activities and a method for producing same by solid state fermentation of wheat bran with Aspergillus niger. 
It is known to produce ethanol from corn starch by an enzymatic method comprising a stage for liquefying the starch with an alpha-amylase for hydrolyzing the starch to dextrins, and then a saccharification stage by a glucoamylase (also called amyloglucosidase) for hydrolyzing the dextrins to glucose, and finally a stage for fermenting the latter to ethanol.
The use of alpha-amylase and glucoamylase enzymes is generally satisfactory when relatively pure starch milk, obtained by the wet milling of corn, is used as starting material, but when it is desired to substitute wheat starches or wheat flours for corn starch, no satisfactory results are obtained with these two enzymes alone because of the presence of hemicelluloses, which increase the viscosity of the saccharified flour worts to the extent that this creates a problem for carrying out the method. It is necessary to use, at the saccharification stage, auxiliary enzymes such as cellulases and hemicellulases in order to reduce the viscosity and remedy this problem. Moreover, it is desirable also to use proteases during saccharification so as to hydrolyze the proteins in the flour and thus enrich the wort with soluble nitrogen in anticipation of the subsequent alcoholic fermentation stage. The traditional supply of nitrogen source necessary for the growth of yeasts during this fermentation may thus be reduced.
All these enzymes are individually commercially available in purified form, but have the disadvantage of being relatively expensive and therefore of increasing the cost of producing ethanol from wheat. In addition, compositions have to be formulated from individual enzymes, which complicates the method.
A need therefore exists for an inexpensive multi-enzyme product combining glucoamylase, proteolytic and hemicellulase activities to produce ethanol from wheat flours at a low cost.
The invention aims to satisfy this need.
The present invention relates to a multi-enzyme product exhibiting glucoamylase, proteolytic and xylanase activities, characterized in that it consists of wheat bran fermented with an Aspergillus niger strain, said enzymatic glucoamylase, proteolytic and xylanase activities being present at the following minimum values:
glucoamylase: at least 100 GU per gram of dry matter,
proteolytic: at least 100 PU per gram of dry matter,
xylanase: at least 100 XU per gram of dry matter, provided that at least one of the following conditions is satisfied:
the glucoamylase activity is at least 750 GU per gram of dry matter
the xylanase activity is at least 300 XU per gram of dry matter.
The glucoamylase activity is preferably at least 1 500 GU per gram of dry matter and/or the xylanase activity is at least 400 XU per gram of dry matter.
Also, the proteolytic activity is preferably at least 400 PU per gram of dry matter.
The invention also relates to a method of producing this multi-enzyme product, characterized in that it comprises the stages consisting in (a) taking wheat bran; (b) moistening and heat-treating said bran so as to pasteurize it or sterilize it; (c) inoculating the resulting wheat bran with an Aspergillus niger strain; (d) the bran being in the form of a layer at least 10 cm thick, fermenting it in the solid state in a reactor which is aerated and stirred periodically for a period of 1 to 3 days, at a temperature of 28-38xc2x0 C., preferably 32 to 36xc2x0 C., said bran being adjusted to an initial moisture content of 50 to 60 wt. % which is substantially maintained during the duration of the fermentation, under aeration conditions appropriate for avoiding accumulation of carbon dioxide which is harmful to the fermentation in the reactor and a rise in temperature due to fermentation above the recommended range, until the fermentation product exhibits the following minimum enzyme activity values:
glucoamylase: at least 100 GU per gram of dry matter,
proteolytic: at least 100 PU per gram of dry matter,
xylanase: at least 100 XU per gram of dry matter, provided that at least one of the following conditions is satisfied:
the glucoamylase activity is at least 750 GU per gram of dry matter
the xylanase activity is at least 300 XU per gram of dry matter.
The glucoamylase activity is preferably at least 1 500 GU per gram of dry matter and/or the xylanase activity is at least 400 XU per gram of dry matter.
Also, the proteolytic activity is preferably at least 400 PU per gram of dry matter.
The Aspergillus niger strain is preferably chosen from the NRRL 3112 strain, the ATCC 76061 strain and the strains obtained from said strains by selection or mutation when a high glucoamylase activity is desired. The ATCC 76061 strain is particularly preferred.
When a high glucoamylase activity is desired, the wheat bran used as starting material should be a non-starch-free bran. Apart from this restriction, any bran may be used. However, the bran preferably comprises a significant proportion (at least 40 wt. %) of particles of less than 1 mm.
The characteristics of two suitable brans are given below by way of illustration.
The wheat bran should be moistened and heat-treated in order to pasteurize it or to sterilize it. It is advantageous that the heat treatment does not precede the moistening because poor fermentation results have been obtained if the bran is heat-treated before moistening. The heat treatment may consist in heating, for example, in an autoclave. An autoclave treatment of 20 min at 120xcx9c121xc2x0 C. has proved highly satisfactory, but less severe conditions (pasteurization at 105xc2x0 C. for 15 min in an oven) are also suitable. It is also possible to carry out the heat treatment of the bran by injecting steam into it, which may make it possible to moisten the bran simultaneously.
The pH may advantageously be adjusted during moistening in the range from 4 to 5.5 in order to improve the pasteurizing effect of the heat treatment and the initiation of the desired fermentation.
In addition to its sterilizing function, the effect of the heat treatment is to promote gelatinization of the starch contained in the wheat bran and therefore the availability of this substrate for the fungus Aspergillus niger, which allows more effective fermentation.
The moistening of the bran is important because the water content influences the performance of the fermentation. The initial water content of the bran is initially adjusted to 50-60%, preferably 50-55%, of the total mass of the bran and of the water and it is substantially maintained in this range during fermentation, for example by periodically supplying water in order to compensate for the loss of water from the medium. The expression xe2x80x9csubstantially maintainedxe2x80x9d means that it is acceptable for the moisture level to take a value which varies slightly (xc2x15% units) from the range 50-60% during a relatively brief period between two successive adjustments of the moisture level or at the end of fermentation. It is advantageous, in any case, not to drop below a moisture level of 45%. The moisture level of the culture medium tends to decrease during the culture through evaporation due to the increase in temperature generated by the fungal growth, said medium being a poor heat conductor. The quality of the water used also plays a significant role. Good quality running water or distilled water may be used.
The inoculation of the wheat bran may be performed with any appropriate inoculum. Persons skilled in the art know many ways of preparing a suitable inoculum from a selected strain. The inoculum dose is advantageously at least 1xc3x97107 spores/gram of initial dry matter.
The fermentation may be carried out in any appropriate reactor. Examples of a reactor which can be used are those described in the paper by A. DURAND et al., published in Agro-Food-Industry Hi-Tech (May-June 1997, pages 39-42).
The fermentation may be carried out for a period of 1 to 3 days, preferably of 30 to 60 hours. At less than 1 day, the fermentation is too incomplete. After 3 days, the fermentation is complete or practically complete and it would be uneconomical to prolong it further. The temperature of the medium is typically maintained between 28 and 38xc2x0 C., preferably between 32 and 36xc2x0 C., which corresponds to the optimum activity range known for the Aspergillus niger strains to be used in the invention. For this purpose, the air temperature is advantageously set at 34-38xc2x0 C. for the first few hours of fermentation in order to promote germination of the spores, and then reduced to 28-32xc2x0 C. for the remainder of the fermentation in order to contribute to the regulation of the temperature of the medium.
The pH of the fermentation medium is not usually regulated. If its starting value is close to 6.0-6.4, the pH decreases to 3.8-4.2 during culture but increases at the end. This change is generally correlated with the fungus sporulation phase. The variation of the pH constitutes a good indicator of the state of the culture.
The fermentor should be aerated, preferably continuously, in order to supply the oxygen necessary for fermentation and to avoid the excessive accumulation of carbon dioxide produced by fermentation. In addition, the aeration helps to control the temperature and the moisture of the culture medium. The air is preferably substantially saturated with water in order to limit the tendency for the medium to dry out. It is difficult to give quantitative information on the aeration rate because many variables, in particular the size and the geometry of the reactor, the quantity of its load, and the like, come into play. Simple routine trials will allow persons skilled in the art to easily determine a suitable aeration rate in each practical case, however.
The bran load in the fermentor should be periodically added during fermentation using stirring means, such as stirring arms, blades or spatulas, or lead screws so as to avoid the formation of impermeable masses and so that the aeration reaches the entire mass of bran as homogeneously as possible. Excessively vigorous stirring which could harm the fungus should be avoided, however.
The product of the invention is a solid product which is useful in particular for the production of ethanol from wheat. It may be directly added to liquefied starch (dextrins) obtained in the liquefaction stage, in order to carry out the saccharification. For this application, it is the glucoamylase activity which is the most important factor. A product of the invention will therefore preferably be used which has a glucoamylase activity of at least 750 GU, for example, preferably of at least 1 500 GU per gram of dry matter.
Another possible use of the product of the invention relates to the production of wheat-based feed for monogastric animals, for example poultry and pigs. In this application, it is the xylanase activity which constitutes the most important factor. A product will therefore be used in this application which preferably has a high xylanase activity, for example of at least 400 XU per gram of dry matter.
The product of the invention may be dried or frozen for storage, if desired.
The drying should be carried out at a moderate temperature so as not to affect the enzyme activity. Heating in an oven at 40xc2x0 C. has proved to be appropriate, for example. Freezing may be carried out on the moist product at low temperature, for example at xe2x88x9220xc2x0 C.
In the examples, the various enzyme activities were measured by the following methods:
a) Glucoamylase activity
The action of a glucoamylase (GA) preparation on a starch solution brings about the release of reducing sugars. Heated at 100xc2x0 C. in the presence of 3,5-dinitrosalicylic acid (DNS), these compositions take on a brown color which is measured on a spectrophotometer (Kontron Instruments, Milan, Italy) at 540 nm.
The reaction medium contains
The reaction occurs for 30 min at 60xc2x0 C. (55xc2x0 C. for the A. orizae GAs). Samples are collected every 5 min, mixed with DNS and placed in an ice bath. They are then heated for 5 min at 100xc2x0 C., rapidly cooled and then assayed at 540 nm.
These assay conditions were established after studying the influence of the temperature and the pH on the activity of the GA preparations. Merck soluble starch (Darmstadt, Germany) was used as substrate for this enzymatic hydrolysis. The DNS is prepared according to the following protocol proposed by P. Bernfeld, Methods in enzymology, 1, 149-159 (1955):
Dissolve beforehand:
10 g of 3,5-dinitrosalicylic acid
200 ml of 2 molar sodium hydroxide
200 ml of distilled water.
Then add:
300 g of sodium potassium tartrate.
Adjust the volume to 1 liter with distilled water after complete dissolution.
Once prepared, this reagent should be stored protected from light. The calibration curves were prepared with glucose as reference product for assaying the glucoamylase activity and for monitoring the liquefaction-saccharification reactions, and with xylose for measuring the xylanase activity.
One glucoamylase activity unit (GU) corresponds to the quantity of enzyme necessary to release one micromole of reducing ends per minute under the assay conditions with glucose as reference. The glucoamylase activity, calculated using the formula indicated below, is expressed relative to the quantity of initial dry matter (IDM):
A=(P/Venz)*(Vferm/Mferm)
A is the GA activity expressed in GU.gIDMxe2x88x921 (xcexcmol.minxe2x88x921.gIDMxe2x88x921),
P is the glucose equivalent release rate in xcexcmol.minxe2x88x921,
Venz is the volume of the enzyme solution assayed in ml,
Vferm is the total volume of distilled water used to extract the enzyme solution in ml,
Mferm, expressed in g of IDM, is the initial mass of dry product from which the enzyme solution was extracted.
b) Protease activity
This assay was developed on azocasein using the Bxc3xa9inon method described in xe2x80x9cProtein Purification Methodsxe2x80x94a Practical Approachxe2x80x9d, Harris E. L. V. and Angal, S (Editors), IRL-Press, Oxford University Press, 1-66 (1989). The degradation of this substrate by proteases causes the release of azo groups which absorb UV at 340 nm. The variation of the absorbence during the kinetics of hydrolysis of this protein indicates the extent of the reaction.
The reaction medium contains:
The azocasein (Sigma, Saint-Louis, United States) is dissolved in a 0.1 M acetate buffer at pH 5.0. The protease activities were assayed at this pH because azocasein is insoluble in this acetate buffer at lower pH values. The enzyme reaction is carried out at 60xc2x0 C. Samples are collected every 5 min for 20 min and mixed with 5% trichloroacetic acid (TCA) to stop the reaction.
One protease activity unit (PU) corresponds to the quantity of enzymes necessary for an increase of 0.01 A340 nm unit per minute, generated by the release of azo groups under the conditions mentioned above. This activity, calculated based on the formula indicated below, is expressed relative to the initial dry matter (PU.Gxe2x88x921 IDM) or the glucoamylase activity (PU.GUxe2x88x921):
A=(P/Venz)*(Vferm/Mferm)
A is the protease activity expressed in PU.gIDMxe2x88x921,
P is the rate of release of the azo groups expressed as an increase of 0.01 unit A340 nm.minxe2x88x921,
Venz is the volume of the enzyme solution assayed in ml,
Vferm is the total volume of distilled water used to extract the enzyme solution in ml,
Mferm, expressed in g of IDM, is the initial mass of dry product from which the enzyme solution was extracted.
c) Xylanase activity
To demonstrate this enzyme activity, the GA preparations are reacted with a soluble xylan solution and the reducing sugars released were measured by the DNS method.
The reaction medium is composed of:
The solution of larch xylan (Sigma at 1%) is prepared in citrate buffer at pH 4.5 and the reaction occurs at 60xc2x0 C. Samples are collected every 5 min for 20 min, mixed with DNS and placed in an ice bath. They are then assayed according to a protocol identical to that presented for the measurement of the GA activities with xylose as reference.
One xylanase activity unit (XU) corresponds to the quantity of enzymes necessary for the release of one micromole of reducing sugars per minute. This activity is expresssed relative to the initial dry matter (XU.gxe2x88x921 IDM) or to the glucoamylase activity (XU.GUxe2x88x921). To calculate this activity, the formula defined for the calculation of the GA activities is used again, in which:
A is the xylanase activity expressed in XU.gIDMxe2x88x921 (xcexcmol.minxe2x88x921.gIDMxe2x88x921),
P is the rate of release of xylose equivalents in xcexcmol.minxe2x88x921,
the other terms of the formula are not modified.
The following nonlimiting examples are given to illustrate the invention.